A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates.
Anal Biochem
; 549: 130-135, 2018 05 15.
Article
en En
| MEDLINE
| ID: mdl-29559333
ABSTRACT
Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
/
Antiportadores
/
Proteínas de Escherichia coli
/
Farmacorresistencia Bacteriana Múltiple
/
Escherichia coli
/
Liposomas
Idioma:
En
Revista:
Anal Biochem
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos